The present invention relates to a method for treatment of restenosis by gene therapy, comprising the administration of a recombinant adenovirus containing a suicide gene. It also relates to particular pharmaceutical compositions permitting the local and effective administration of recombinant viruses.
Atherosclerosis is a complex, polygenic disease which is defined in histological terms by deposits (lipid or fibrolipid plaques) of lipids and of other blood derivatives in the wall of the large arteries (aorta, coronary arteries, carotid). These plaques, which are more or less calcified according to the degree of progression of the process, may be coupled with lesions and are associated with the accumulation in the arteries of fatty deposits consisting essentially of cholesterol esters. These plaques are accompanied by a thickening of the arterial wall, with hypertrophy of the smooth muscle, appearance of foam cells and accumulation of fibrous tissue. The atheromatous plaque protrudes markedly from the wall, endowing it with a stenosing character responsible for vascular occlusions by atheroma, thrombosis or embolism, which occur in those patients who are most affected. These lesions can hence lead to very serious cardiovascular pathologies such as infarction, sudden death, cardiac insufficiency, stroke, and the like.
Since 1977, the technique of angioplasty has been developed to permit a non-surgical intervention in respect of the atherosclerosis plaque. However, the treatment of an atherosclerotic lesion by angioplasty results very frequently (up to 50% of cases in some studies) in a restenosis following mechanical injury of the arterial wall. A key event in this mechanism is the proliferation and migration of vascular smooth muscle cells (VSMC) from the media to the intima, in particular as a result of the absence of protection and/or feedback control exercised by the endothelial cells of the intima.
The treatment of restenosis by administration of chemical or proteinaceous substances capable of killing vascular smooth muscle cells has been proposed in the prior art. Thus, psolaren derivatives, incorporated by proliferative cells and then sensitizing these cells to the action of light, have been used (March et al., 1993, circulation, 87:184-191). Similarly, some cytotoxins consisting of a fusion protein between a plant or bacterial toxin fragment and a growth factor have also been used (Pickering et al., J. Clin. Invest., 1993, 91:724-729; Biro et al., 1992, Circ. Res., 71:640-645; Casscells et al., Proc. Natl. Acad. Sci. USA, 1992, 89:7159-7163). However, these treatments have many drawbacks, such as their low specificity, their indifferent efficacy, a considerable delay in acting and a potential toxicity.
The present invention offers an advantageous approach to this problem. The present invention provides, in effect, an especially effective and selective method for the treatment of postangioplasty restenosis by gene therapy. The method of the present invention consists mainly in administering a recombinant adenovirus containing a suicide gene, capable of specifically sensitizing proliferating vascular smooth muscle cells to a therapeutic agent. Simultaneous or subsequent administration of this therapeutic agent then brings about the selective death of the sensitized cells.
The advantages of the present invention lie, in particular, in the high capacity of the adenoviruses of the invention to infect proliferating vascular smooth muscle cells. This enables relatively small amounts of active principle (recombinant adenovirus) to be used, and also permits an effective and very rapid action on the sites to be treated. The adenoviruses of the invention are also capable of expressing at very high levels the suicide genes introduced, thereby endowing them with a very effective therapeutic action. Furthermore, on account of their episomal character, the adenoviruses of the invention have a limited persistence in proliferative cells, and hence a transient effect entirely suited to the desired therapeutic effect. Lastly, the Applicant has also developed an especially advantageous method of administration, which enables certain target cells essential to the desired therapeutic effect to be infected with great efficacy.
A first object of the invention hence relates to the use of a defective recombinant adenovirus containing a suicide gene for the preparation of a pharmaceutical composition intended for the treatment of restenosis.
As mentioned above, for the purposes of the present invention, suicide gene is understood to mean any gene whose expression product endows the infected cell with a sensitivity to a therapeutic agent. As an example, there may be mentioned the thymidine kinase gene, whose expression product endows mammalian cells with a sensitivity to certain therapeutic agents such as ganciclovir or acyclovir, or the cytosine deaminase gene, whose expression product endows mammalian cells with a sensitivity to 5-fluorocytosine (5-FC).
Herpes simplex virus thymidine kinase is capable of phosphorylating nucleoside analogues such as acyclovir and ganciclovir. These modified molecules may be incorporated in a DNA chain undergoing elongation, which results in the cessation of DNA synthesis and brings about the death of the cell (F. L. Moolten, Cancer Res. 46 (1986) 5276). This strategy thus enables cells expressing the TK gene to be specifically eliminated. Furthermore, since the DNA synthesis is the target of the toxicity, only cells undergoing division are affected.
More preferably, the human herpesvirus thymidine kinase (hHSV-1 TK) gene is used in the context of the present invention. The sequence of this gene has been described in the literature (see, in particular, McKnight et al., Nucleic Acid. Res. 8 (1980) 5931). It is also possible to use derivatives of this sequence displaying greater substrate specificity or better kinase activity. Such derivatives may, in particular, be obtained by mutagenesis at the binding site, as described previously (Balasubramaniam et al., J. Gen. Virol. 71 (1990) 2979; Munir et al., JBC 267 (1992) 6584).
It is also possible to use the cytosine deaminase gene, whose expression product endows mammalian cells with a sensitivity to 5-fluorocytosine (5-FC). Cytosine deaminase is capable of catalyzing the deamination of cytosine to uracil. Cells which express this gene are hence capable of converting 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), which is a toxic metabolite. The sequence of this gene has been described in the literature (Anderson et al., Arch. Microbiol. 152 (1989) 115).
More generally, any gene capable of endowing infected cells with a sensitivity to a therapeutic agent may be used in the context of the present invention. The thymidine kinase gene constitutes an especially advantageous embodiment.
For the construction of the adenoviruses according to the invention, different serotypes may be used. There are, in effect, many serotypes of adenovirus, whose structure and properties vary somewhat. Among these serotypes, it is preferable however to use, in the context of the present invention, human adenoviruses type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see Application FR 93/05954). Among adenoviruses of animal origin which are usable in the context of the present invention, adenoviruses of canine, bovine, murine (for example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or alternatively simian (for example: SAV) origin may be mentioned. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan or A26/61 (ATCC VR-800) strain, for example]. It is preferable to use adenoviruses of human or canine or mixed origin in the context of the invention.
As stated above, the adenoviruses according to the invention are defective, that is to say they are incapable of replicating autonomously in the target cell. Generally, the genome of the defective adenoviruses used the context of the present invention hence lacks at least the sequences needed for replication of the said virus in the infected cell. These regions may be either removed (in their entirety or partially), or rendered non-functional, or replaced by other sequences, and in particular by the suicide gene. Preferably, the defective adenovirus nevertheless retains the sequences of its genome which are needed for encapsidation of the viral particles.
Preferably, the defective adenoviruses of the invention comprise the ITRs, a sequence permitting encapsidation and the suicide gene. Still more preferably, in the genome of the adenoviruses of the invention, the E1 gene and at least one of the genes E2, E4, L1-L5 are non-functional. The viral gene of interest may be rendered non-functional by any technique known to a person skilled in the art, and in particular by total elimination, substitution, partial deletion or addition of one or more bases in the gene or genes of interest. Such modifications may be obtained in vitro (on the isolated DNA) or in situ, for example by means of genetic engineering techniques, or alternatively by treatment using mutagenic agents.
More preferably, a defective adenovirus rendered non-functional by a total or partial deletion of the E1 region and a deletion in the E4 region is used. The E4 region comprises 7 reading frames. The deletion in the E4 region may be transcomplemented by the presence, in the cell line used for multiplication of the viruses, either simply of the reading frame ORF6, or of the reading frames ORF6 and ORF6/7.
Preferred adenoviruses according to the invention are chosen from the following:
Defective recombinant adenovirus xcex94E1, xcex94E4 comprising a deletion of all or part of the E1 region and a deletion of all or part of the E4 region.
Defective recombinant adenovirus xcex94E1, ORF3xe2x88x92, ORF6xe2x88x92 comprising a deletion of all or part of the E1 region and of nucleotides 34801-34329 and 34115-33126 of the E4 region.
Defective recombinant adenovirus xcex94E1, xcex94E4, ORF1+ comprising a deletion of all or part of the E1 region and a deletion of the E4 region except for the reading frame ORF1. More specifically, the deletion in the E4 region has its 5xe2x80x2 end included in the reading frame ORF7 and its 3xe2x80x2 end included in the reading frame ORF2. For example, in the region covering nucleotides 33093-35053.
Defective recombinant adenovirus xcex94E1, xcex94E4, ORF4+ comprising a deletion of all or part of the E1 region and a deletion of the E4 region except for the reading frame ORF4. More especially, two deletions are carried out, one whose 5xe2x80x2 end is included in the reading frame ORF7 and whose 3xe2x80x2 end is located in the reading frame ORF6, the other whose 5xe2x80x2 end is included in the reading frame ORF3 and whose 3xe2x80x2 end is located in the reading frame ORF1 or in the promoter region of E4. For example, a deletion covering nucleotides 33093-33695 and a deletion covering nucleotides 34634-35355.
Defective recombinant adenovirus xcex94E1, xcex94E4 comprising a deletion of all or part of the E1 region and a deletion covering the whole of the E4 region, chosen, for example, from the following deletions: nucleotides 32720-35835, or 33466-35355, or 33093-35355.
The construction of these vectors is described in Patents No. FR 9500749 and No. FR 9506532.
The defective recombinant adenoviruses according to the invention may be prepared by any technique known to a person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185,573; Graham, EMBO J. 3 (1984) 2917). In particular, they may be prepared by homologous recombination between an adenovirus and a plasmid carrying, inter alia, the suicide gene. Homologous recombination takes place after cotransfection of the said adenovirus and said plasmid into a suitable cell line. The cell line used should preferably (i) be transformable by the said elements, and (ii) contain the sequences capable of complementing the portion of the genome of the defective adenovirus, preferably in integrated form in order to avoid risks of recombination. As an example of a line, there may be mentioned the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains, in particular, integrated in its genome, the left-hand portion of the genome of an Ad5 adenovirus (12%). Strategies of construction of vectors derived from adenoviruses have also been described in Applications Nos. FR 93/05954 and FR 93/08596.
Thereafter, the adenoviruses which have multiplied are recovered and purified according to standard techniques of molecular biology, as illustrated in the examples.
Advantageously, in the adenoviruses of the invention, the suicide gene is placed under the control of a promoter permitting its expression in infected cells. This promoter can be the one belonging to the suicide gene, a heterologous promoter or a synthetic promoter. In particular, promoters originating from eukaryotic or viral genes may be used. For example, it is possible to use promoter sequences originating from the genome of the cell which it is desired to infect. Similarly, the promoter sequences may originate from the genome of a virus, including that of the virus used. In this connection, the E1A, MLP, CMV, RSV LTR, and the like, promoters may, for example be mentioned. In addition, these expression sequences may be modified by adding activation or regulatory sequences or sequences permitting a tissue-specific expression. It can, in effect, be especially advantageous to use expression signals which are active specifically or predominantly in vascular smooth muscle cells, so that the suicide gene is expressed and produces its effect only when the virus has actually infected a vascular smooth muscle cell. Among promoters which are active specifically or predominantly in vascular smooth muscle cells, the promoter of xcex1-actin of smooth muscle may be mentioned in particular.
In a particular embodiment of the invention, a defective recombinant adenovirus is used which comprises a suicide gene under the control of a viral promoter, preferably chosen from the RSV LTR and the CMV early promoter.
According to another advantageous embodiment, the promoter used is one which is active specifically or predominantly in vascular smooth muscle cells.
The present invention thus provides an especially effective method for the treatment of restenosis. Moreover, to increase further the efficacy and specificity of the treatment, the Applicant has developed a method permitting a local administration of the recombinant adenoviruses at the sites to be treated. More especially, this method is based on the use of an angioplasty balloon coated with a hydrophilic film (for example a hydrogel) impregnated with adenovirus, which may thus be applied precisely to the site to be treated and permit a local and effective release of the adenoviruses at the cells to be treated.
In addition, the Applicant showed that, on healthy arteries, this method of administration made it possible to infect a high percentage of cells of the media (up to 9.6%), which are the most logical targets for the prevention of restenosis.
In a most particularly advantageous aspect, the Applicant also showed that the virus and the method of the invention permitted an effective and selective transfer of genes into an atheromatous artery. More especially, the Applicant has demonstrated for the first time the capacity of adenoviruses to transfer a therapeutically effective gene into an atheromatous artery. This is absolutely essential, since the therapeutic efficacy of the treatment of restenosis is conditional on a demonstration of the capacity to transfer the therapeutic gene, into the correct cells and with suitable efficacy, under the physiopathological conditions. Atheromatous arteries are characterized by the presence in the intima (i) of deposits of extracellular matrix, (ii) of lipid deposits consisting essentially of foam cells of the macrophage type and (iii) of proliferating smooth muscle cells.
The results presented below show that, in these atheromatous arteries, the viruses according to the invention permit a lower percentage of infection but which is of greater specificity (taking account, in fact, of the presence of macrophage type cells in this case, macrophage cells not being transduced) and which is accompanied by a substantial therapeutic efficacy. The results obtained show, in particular, a very selective transfer of the adenovirus into the target cells, that is to say the proliferating smooth muscle cells. Out of the whole cell population present in the atheromatous zone, more than 95% of the infected cells are vascular smooth muscle cells. Thus, the macrophage cells present in the intima are not infected at all (no infected macrophage cell was detected). As regards the proliferating smooth muscle cells (in the neointima), the treatment according to the invention enables a percentage of less than 1% (for example 0.2%) to be infected. This is much less than the results described previously in healthy arteries or those possessing lesions of the wall but which do not represent a physiopatholigical situation of restenosis (endothelial abrasion of a healthy artery). The Applicant also showed that the infection of this small percentage of cells nevertheless permitted a substantial therapeutic effect, demonstrated, in particular, by measurement of the luminal diameter. This result is especially surprising and implies the existence of an induced cytotoxic effect (xe2x80x9cbystanderxe2x80x9d effect) in vivo. Hence the invention describes for the first time a method permitting the selective transfer of genes into proliferating vascular smooth muscle cells in an atheromatous artery, comprising the administration into the said artery of a defective recombinant adenovirus containing the said gene by means of an angioplasty balloon catheter. The term selective transfer implies a transfer essentially into the proliferating vascular smooth muscle cells and no transfer into the surrounding macrophage cells. This method permits a treatment of restenosis by transfer of a suicide gene such as the TK gene, followed by treatment with ganciclovir or acyclovir, for example. This method of treatment is, in addition, characterized by an effect of toxicity induced in vivo.
Another subject of the present invention relates to a pharmaceutical composition comprising a defective recombinant adenovirus and a hydrogel. More specifically, the invention relates to a composition comprising a defective recombinant adenovirus containing a suicide gene, and a hydrogel. The hydrogel used in the context of the present invention may be prepared from any biocompatible and non-cytotoxic (homo- or co-)polymer. Such polymers have, for example, been described in Application WO93/08845. Some of them, such as, in particular, those obtained from ethylene oxide and/or propylene oxide, are commercially available.
The method of treatment of the invention hence advantageously consists in introducing, at the site to be treated, a composition comprising a hydrogel impregnated with recombinant adenoviruses. The hydrogel may be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Advantageously, the hydrogel [lacuna] be introduced into the site to be treated by means of a catheter, for example a balloon catheter, in particular during angioplasty, thereby enabling any additional trauma due to a further intervention at the angioplasty site to be avoided. It is especially advantageous for the impregnated hydrogel to be introduced into the site to be treated by means of a balloon catheter protected by a sleeve. As described in the examples, the hydrogel possesses a large number of advantages: it enables sliding of the balloon to be improved, thereby enabling it to pass through heavily stenosed arteries. Furthermore, the hydrogel can be used with any type of angioplasty balloon, which enables perfusion balloons to be used in particular. Thus, according to a particular embodiment, the adenoviruses according to the invention are administered by means of perfusion balloons, especially channelled balloon catheters (xe2x80x9cchannelled balloon angioplasty catheterxe2x80x9d, Mansfield Mecical, Boston Scientific Corp., Watertown, Mass.). The latter consists of a conventional balloon covered with a layer of 24 perforated channels which are perfused via an independent lumen through an additional infusion orifice. These perfusion balloons, which make it possible to maintain a blood flow and thus to decrease the risks of ischaemia of the myocardium, on inflation of the balloon, also enable a medicinal product to be delivered locally at normal pressure for a relatively long time, more than twenty minutes, which is necessary for an optimal infection.
It is especially advantageous to use a perfusion balloon catheter coated with hydrogel. In this case, the advantages of both, that is to say the possibility of keeping the balloon inflated for a longer period of time by retaining the properties of facilitated sliding and of site-specificity of the hydrogel, are gained simultaneously. In this case, an optimal efficacy of infection is obtained.
The results presented in the examples demonstrate, in fact, the efficacy of this system for the percutaneous transfer of genes into the arterial walls.
Another subject of the present invention relates to a pharmaceutical composition comprising a defective recombinant adenovirus and poloxamer. More specifically, the invention relates to a composition comprising a defective recombinant adenovirus containing a suicide gene, and poloxamer. Poloxamer 407 is a non-toxic, biocompatible polyol; it is commercially available (BASF, Parsippany, N.J.).
Hence a method of treatment of the invention advantageously consists in introducing, into the site to be treated, a composition comprising poloxamer impregnated with recombinant adenoviruses. The poloxamer may be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Advantageously, the poloxamer may be introduced into the site to be treated by means of a catheter, for example a balloon catheter, in particular during angioplasty, thereby enabling any additional trauma due to a further intervention at the angioplasty site to be avoided. It is especially advantageous for the impregnated poloxamer to be introduced into the site to be treated by means of a balloon catheter protected by a sleeve. Poloxamer possesses essentially the same advantages as hydrogel while having a lower viscosity.
It is especially advantageous to use a perfusion balloon catheter coated with poloxamer, especially channelled balloon catheters. In this case, the advantages of both, that is to say the possibility of keeping the balloon inflated for a longer period of time while retaining the properties of facilitated sliding and of site-specificity of the poloxamer, are gained simultaneously. In this case also, an optimal efficacy of infection is obtained.
The present invention will be described more completely by means of the examples which follow, which are to be considered to be illustrative and non-limiting.